JGV Papers in Press. Published March 11, 2015 as doi:10.1099/vir.0.000120
Journal of General Virology CD4 binding site broadly neutralizing antibody selection of HIV-1 escape mutants --Manuscript Draft-Manuscript Number:
VIR-D-15-00017R2
Full Title:
CD4 binding site broadly neutralizing antibody selection of HIV-1 escape mutants
Short Title:
selection of HIV-1 neutralization escape mutants
Article Type:
Short Communication
Section/Category:
Animal - Retroviruses
Corresponding Author:
Aine McKnight Queen Mary's School of Medicine and Dentistry London, UNITED KINGDOM
First Author:
Hanna Dreja
Order of Authors:
Hanna Dreja Corinna Pade Lei Chen Aine McKnight
Abstract:
All human-immunodeficiency-virus type-1 (HIV-1) viruses use CD4 to enter cells. Consequently the viral envelope CD4-binding-site (CD4bs) is relatively conserved, making it a logical neutralizing antibody target. It is important to understand how CD4binding site variation allows for escape from neutralizing antibodies. Alanine scanning mutagenesis identifies residues in antigenic sites, whereas escape mutant selectionI dentifies viable mutants. We selected HIV-1 to escape CD4bs neutralizing MAbs b12, A12 and HJ16. Viruses that escape from A12- and b12- remained susceptible to HJ16, VRC01 and J3, whilst six different viruses that escape HJ16 remained sensitive to A12, b12 and J3. In contrast, their sensitivity to VRC01 was variable. Triple HJ16/A12/b12resistant virus proved that HIV-1 can escape multiple BNMAbs, but still retain sensitivity to VRC01 and the llama derived J3 nanobody. This antigenic variability could reflect that occurring in circulating viruses so studies like this could predict immunologically relevant antigenic forms of the CD4bs for inclusion in HIV-1 vaccines.
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CD4 binding site broadly neutralizing antibody selection of HIV-1 escape mutants
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Hanna Dreja1, Corinna Pade1, Lei Chen2, Áine McKnight*1
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Centre for Immunology and Infectious Disease, Blizard Institute, Barts & The London School of Medicine and Dentistry, Queen Mary University of London 2
Vaccine Research Center, NIAID, National Institutes of Health, Bethesda, Maryland
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Running Title: Neutralizing antibody selection of HIV-1 escape mutants
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*Corresponding Author: Áine McKnight email [email protected]
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Words 2229
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Abstract
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All human-immunodeficiency-virus type-1 (HIV-1) viruses use CD4 to enter cells. Consequently the
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viral envelope CD4-binding-site (CD4bs) is relatively conserved, making it a logical neutralizing
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antibody target. It is important to understand how CD4-binding site variation allows for escape
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from neutralizing antibodies. Alanine scanning mutagenesis identifies residues in antigenic sites,
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whereas escape mutant selection identifies viable mutants. We selected HIV-1 to escape CD4bs
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neutralizing MAbs b12, A12 and HJ16. Viruses that escape from A12- and b12- remained
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susceptible to HJ16, VRC01 and J3, whilst six different viruses that escape HJ16 remained sensitive
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to A12, b12 and J3. In contrast, their sensitivity to VRC01 was variable. Triple HJ16/A12/b12-
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resistant virus proved that HIV-1 can escape multiple BNMAbs, but still retain sensitivity to VRC01
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and the llama derived J3 nanobody. This antigenic variability could reflect that occurring in
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circulating viruses so studies like this could predict immunologically relevant antigenic forms of the
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CD4bs for inclusion in HIV-1 vaccines.
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A successful human immunodeficiency virus type 1 (HIV-1) vaccine is expected to need to induce
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robust CD4+ and CD8+ cellular responses, in concert with a strong and broadly neutralizing
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antibody response. Designing immunogens that trigger such responses is challenging (reviewed in
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(Haynes & Montefiori, 2006; McCoy & Weiss, 2013)), partly due to the diversity (Gaschen et al.,
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2002) of the viral envelope glycoprotein (Env), which interacts with cell receptors including CD4.
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Hence, the CD4 binding site (CD4bs) is functionally conserved and is therefore a logical vaccine
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target to elicit neutralizing Abs. A number of highly effective anti-CD4bs broadly neutralizing
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monoclonal antibodies (BNMAbs) with distinct neutralization profiles have been generated from
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HIV-1 infected individuals (Burton et al., 1991; Burton et al., 1994; Corti et al., 2010; Falkowska et
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al., 2012; Walker et al., 2009; Wu et al., 2010; Zhou et al., 2010) or vaccinated llamas (Forsman et
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al., 2008; McCoy et al., 2012). A full understanding of the nature of these antibodies would
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provide clues to the antigenic landscape of the CD4bs, which may be important for developing an
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inclusive HIV-1 vaccine. In addition to structurally define current BNMAbs and their corresponding
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CD4bs footprints, the ability of replication competent viruses to escape such antibodies and to
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determine whether the mutants remain susceptible to alternative anti-CD4bs BNMAbs will be
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valuable.
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We used the three different anti-CD4bs BNMAbs b12, A12 and HJ16 to select for escape mutants.
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B12 was the first human BNMAb to map to the CD4bs and competes for binding to soluble CD4
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(sCD4) (Barbas et al., 1992; Burton et al., 1991; Burton et al., 1994; Roben et al., 1994; Zhou et al.,
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2010). The llama-derived single chain Ab A12 also competes with b12 and sCD4 (Forsman et al.,
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2008), as does HJ16 (Corti et al., 2010). Alanine scanning has determined that HJ16 belongs to a
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class of antibodies, which binds to a region distinct from the classic CD4bs (AA 474-476) (Pietzsch
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et al., 2010). Recently, Balla-Jhagjhoorsingh et al (Balla-Jhagjhoorsingh et al., 2013) identified a
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glycosylation site (N276) critical for HJ16-induced escape of a primary HIV-1 strain in an in vivo
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model.
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We used the well described HIV-1 replication competent clone HXB2 (Ratner et al., 1985) as it is
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unlike primary isolates highly sensitive to many anti-CD4bs BNMAbs. This is probably because
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HXB2 was highly passaged in vitro in the absence of humoral responses Escape viruses were
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selected in C8166 CD4+ T-cells (Salahuddin et al., 1983) in gradually increasing concentrations of
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BNMAbs (from 50 ng NMAb ml-1). Cell free supernatants were harvested from cells with cytopathic
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appearances and added to target cells for a second round of infection, this time with a doubling of
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the BNMAb concentration. After two to four weeks, resistant viruses emerged that could replicate
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in the presence of high concentrations of each BNMAb (10 µg A12 or HJ16 ml–1 and 20 µg b12 ml–
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resistant viruses were PCR amplified and sequenced (Dreja et al., 2010).
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Selection of HXB2 with b12 resulted in a virus with the single dominant AA change (G366E) within
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the envelope CD4bs (Fig 1, 3). This change is located three AAs upstream of the proline to lysine
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mutation seen in a previous escape study (Mo et al, 1997), where additionally two mutations were
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observed in the V2 region. Interestingly, we did not identify any additional changes. This lack of
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other mutations within the V2 could be explained by the ease of neutralisation of HXB2,
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suggesting that supplementary V2 compensatory mutations were not required for resistance. It
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may be that the G366E mutation did not hamper viral infectivity. Glycine 366 has previously been
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implicated in b12 binding (Li et al., 2011; Saphire et al., 2001, Zhou et al., 2007). The site is
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mapped to a model of the crystal structure of the trimeric HIV-1 Env spike, (Fig 3). The HIV-1 viral
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env-expression vector psvIII-HXB2 (Gao et al., 1996) was engineered by site-directed mutagenesis
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(SDM) to carry the G366E mutation. SDM-pseudotyped virions carrying the luciferase reporter
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gene were produced as previously described (Dreja et al., 2010). The resulting SDM(b12)
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confirmed the b12 resistant phenotype (Fig 2a). The mutation had no effect on viral susceptibility
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to A12 and HJ16, even though both these BNMAbs compete for binding with b12, with each other
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and with sCD4. Moreover the sensitivity of SDM(b12) to VRC01 (Wu et al., 2010; Zhou et al., 2010)
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remained the same (Fig 2b). The recently described llama derived anti-CD4 BNMAb J3 (McCoy et
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al., 2012) neutralized SDM(b12) as efficiently as wild type (WT)-HXB2 (Fig 2b). These results
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suggest that the AA change responsible for b12 escape is antigenically distinct from those of the
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human BNMAbs HJ16 and VRC01 and the llama BNMAbs A12 and J3. The viral escape from b12
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had little effect on the ability of virus to replicate in vitro (data not shown) but was measurably
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less sensitive to CD4-IgG2 inhibition (IC50 shifted from 5ng ml-1 to 50 ng ml-1 and for the
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pseudotype SDM(b12) from 10ng ml-1 to 100 ng ml-1, Fig 2b).
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The A12-selected escape virus was resistant to A12 and carried the S375N mutation adjacent to
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the CD4bs (Fig 1 and 3). SDM(A12) confirmed that A12 resistance is conferred entirely by this
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mutation (Fig 2a). SDM(A12) remained sensitive to both b12 and HJ16, supporting the notion that
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the epitopes of A12 and b12 or HJ16 are distinct. The J3 llama antibody neutralized SDM(A12) and
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there is a small reduction in sensitivity to VRC01 for SDM(A12). There was, however, no apparent
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effect on the sensitivity of the mutation in SDM(A12) to CD4-IgG2.
). Neutralization assays were carried out and proviral full length env from cells infected with
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In contrast to the b12 and A12 selection, where dominant genotypes were generated, selection
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with HJ16 yielded a viral swarm, containing several different mutant viruses. It could be that there
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are more options for escape routes with this single chain antibody compared to bivalent
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antibodies which may have more steric hindrance. The proviral env sequences amplified from HJ16
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viral selected cultures (from nucleotide 127 (KpnI) to 2251 (BamHI)) were inserted into the env-
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expression vector psvIII-HXB2 and six different infectious, resistant clones were identified.
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Interestingly, and in keeping with the observation that glycosylation may be associated with HJ16
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resistance in primary cell cultures (Balla-Jhagjhoorsingh et al., 2013), we also observed that four
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mutations of a potential N-linked glycosylation site in the V5 loop affected sensitivity to HJ16
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neutralization (N463S, S465F, S465P and S465Y) (Fig 1, 3). Gray et al, (Gray et al., 2011)
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demonstrated a relatively high degree of sequence variation within the V5 loop in a large,
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independent panel of Envs, which may affect the accessibility to the CD4bs. Remarkably, none of
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these four substitutions significantly affected CD4-IgG2 inhibition (Fig 2b). The mutations are
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eleven and nine AAs upstream of the core region (474-476), identified as a HJ16 target by Pietzsch
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et al (Pietzsch et al., 2010). Curiously, of the three HJ16 resistant pseudoviruses with substitutions
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at position 465, two (psHJ16(S465F) and psHJ16(S465P)) gained sensitivity to VCR01 (Fig 2a). This
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is concurrent with alanine substitution of this residue (Falkowska et al., 2012), which increased
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sensitivity to VRC01 neutralization. In contrast, psHJ16(S456Y) retained wildtype sensitivity to
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VRC01. This suggests that the glycosylation per se is not important for the antibody footprint of
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VRC01, although it appears crucial for HJ16 activity. Similarly, psHJ16(N463S) maintained wildtype
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sensitive to VRC01. Overall our results suggest that the V5 region is involved in HJ16 and VRC01
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binding, as changes in this domain affect neutralization to both BNMAbs. The fifth HJ16 resistant
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virus had a glycine to aspartic acid change at position 459 (psHJ16D(G459D)), and resulted in a
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virus that was marginally more resistant to VRC01. This mutation was identified in HIV-1 (JRCSF)
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infected humanised mice treated with 45-46G54W, a BNMAb belonging to the VRC01 family (Klein
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et al, 2012). The G459D mutation is only four AAs upstream of the glycosylation site in the V5 loop,
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and exhibits a similar neutralisation profile as psHJ16(N463S) . By contrast, similar to S465F and
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S465P, the HJ16 resistant clone E409R also became more neutralization sensitive to VRC01 (Fig
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2b). All HJ16 resistant pseudotyped viruses retained sensitivity to J3, b12, CD4-IgG2 and, in four
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cases, to A12. Interestingly, G459D and E409R appeared more sensitive to A12 neutralisation at
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lower concentrations (1000 ng x mL-1). This partial VRC01 resistance is in accordance with the findings from
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the individual AA changes, where the N463S and G366E mutations were neutral, whereas S375N
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renders the virus less sensitive to VRC01 neutralization (Fig 2b). SDM(HJ16/A12/b12) was
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marginally less sensitive to J3 (IC50 from 3 to 25 ng x mL-1), as predicted by the lower sensitivity of
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the S375N and G366E mutations (Fig 2b). As expected, the sensitivity to CD4-IgG was partly lost,
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which is likely to be due to the G366E mutation described above (IC50 from 2 to 20 ng x mL-1).
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Nevertheless, it is intriguing that albeit marginally resistant to the BNMAbs VRC01 and J3, the
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triply selected virus retains some sensitivity to these BNMAbs. This suggests that if one succeeds in
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inducing a range of different anti-CD4bs Abs by vaccination, neutralization control of HIV-1 may be
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achieved. Also, the three BNMAbs, provided at the same time, never enabled for a resistant virus
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to emerge, cautiously suggesting that there is a limit to the amount of pressure the virus can
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withstand. Pre-exposure prophylaxis using modified BNMAb is considered as an option to prevent
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HIV-1 acquisition (Pace et al., 2013). Our findings would support such an approach and suggest
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that multiple BNMAbs targeting the CD4bs structure should be considered.
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Studies of the antigenic landscape of BNMAbs and the escape routes viruses master can help us to
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target important immunogenic epitopes for HIV-1 vaccines, but also to consider including
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anticipated escape structures.
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Authors' contributions
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HD and AM conceived and designed the experiments. HD and CP acquired the data. HD and AM
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interpreted the data and drafted the manuscript. LC mapped mutations onto HIV-1 trimer and
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provided structural context to data. All authors read and approved the final manuscript.
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Acknowledgements
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B12 was provided by Dennis Burton, The Scripps Research Institute; A12 and J3 from Robin Weiss,
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UCL; HJ16 from Humabs Biomed SA, Switzerland. VRC01 was obtained through the AIDS Research
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and Reference Reagent Program, Division of AIDS, NIAID, NIH, from John Mascola and CD4-IgG2
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through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Cat#11780 from Progenics
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Pharmaceuticals. The genetic replication competent clone HXB2 was obtained from the Centre for
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AIDS Research (NIBSC, UK). psvIII-HXB2 was provided by Paul Clapham, Worcester, MA. This
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research was conducted as part of the Collaboration for AIDS Vaccine Discovery funded by the Bill
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and Melinda Gates Foundation (Weiss VDAC, UCL). We would like to thank Robin Weiss, Peter
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Kwong and Laura McCoy for critically reading the manuscript.
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Figure legends
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Figure 1
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Sequences of the BNMAbs-selected EMs are compared to the parental HXB2 env gene, with the nucleotide number in italics to the left. EM3 is the HJ16/A12/b12 triply selected virus.
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Figure 2
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HXB2 (white discs), SDM(A12), SDM(b12) and HJ16-resistant pseudotyped HIV-1 virions (black discs) were assessed for neutralization resistance against against (a) A12, b12 and HJ16 and (b) VRC01, J3 and CD4-IgG2. The % of neutralization is shown on the y-axis in the presence of BNMAbs (in bold at the bottom of the graph) at different concentrations (x-axis: ng x ml-1).
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Figure 3
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A model of the crystal structure of the HIV-1 Env trimer, where identified AA changes are indicated with arrows. HXB2 gp120 is adapted from 3JWD (Pancera et al., 2010). The trimer is adapted from 4NCO.
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Figure Click here to download Figure: Dreja 10 Feb2015.pptx
Figure 1
HXB2
360 FKQS SGGDPE IV THSFNCGG E FFYCNSTQL FNSTWFNST W STEGSNNT EG SD TITLPCRI KQIIN MWQKV GKA MYAPPIS
GQ IRCSSNIT G LLLTRDGGN SNNESEIFR P
EM(b12)
---- -E---- -- -------- - --------- --------- - -------- -- -- -------- ----- ----- --- ------- -- -------- - --------- --------- -
EM(A12)
---- ------ -- --N----- - --------- --------- - -------- -- -- -------- ----- ----- --- ------- -- -------- - --------- --------- -
EM(HJ16F) ---- ------ -- -------- - --------- --------- - -------- -- -- -------- ----- ----- --- ------- -- -------- - --------- ----F---- EM(HJ16P) ---- ------ -- -------- - --------- --------- - -------- -- -- -------- ----- ----- --- ------- -- -------- - --------- ----P---- EM(HJ16Y) ---- ------ -- -------- - --------- --------- - -------- -- -- -------- ----- ----- --- ------- -- -------- - --------- ----Y---- EM(HJ16S) ---- ------ -- -------- - --------- --------- - -------- -- -- -------- ----- ----- --- ------- -- -------- - --------- --S------ EM(HJ16D) ---- ------ -- -------- - --------- --------- - -------- -- -- -------- ----- ----- --- ------- -- -------- - -------D- --------- -
EM(HJ16R) ---- ------ -- -------- - --------- --------- - -------- R- -- -------- ----- ----- --- ------- -- -------- - --------- --------- EM3
---- -E---- -- --N----- - --------- --------- - -------- -- -- -------- ----- ----- --- ------- -- -------- - --------- --S------ -
psHJ16 (S465Y)
psHJ16 (N463S)
psHJ16 (G459D)
psHJ16 (E409R)
% neutralization % neutralization % neutralization
psHJ16 (S465P)
% neutralization
psHJ16F (S465F)
% neutralization
SDM(b12) G366E
% neutralization
WT HXB2
% neutralization
SDM(A12) S375N
% neutralization
Figure 2A 120 100 80 60 40 20 0 120 100 80 60 40 20 0 120 100 80 60 40 20 0 120 100 80 60 40 20 0 120 100 80 60 40 20 0 120 100 80 60 40 20 0 120 100 80 60 40 20 0 -20 120 100 80 60 40 20 0 -20
A12
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b12 120 100 80 60 40 20 0 120 100 1000 100 80 60 40 20 0 100 1000 120
100 80 60 40 20 0 120 10 100 1000 100 80 60 40 20 0 120 10 100 1000 100 80 60 40 20 0 10 100 1000120 100 80 60 40 20 0 10 100 1000 120 100 80 60 40 20 0 10 100 1000120 100 80 60 40 20 ng x ml-1 0 10
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HJ16: N463S HJ16: G459D HJ16: E409R
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CD4BS A12: S375N
Journal of General Virology CD4 binding site broadly neutralizing antibody selection of HIV-1 escape mutants --Manuscript Draft-Manuscript Number:
VIR-D-15-00017R2
Full Title:
CD4 binding site broadly neutralizing antibody selection of HIV-1 escape mutants
Short Title:
selection of HIV-1 neutralization escape mutants
Article Type:
Short Communication
Section/Category:
Animal - Retroviruses
Corresponding Author:
Aine McKnight Queen Mary's School of Medicine and Dentistry London, UNITED KINGDOM
First Author:
Hanna Dreja
Order of Authors:
Hanna Dreja Corinna Pade Lei Chen Aine McKnight
Abstract:
All human-immunodeficiency-virus type-1 (HIV-1) viruses use CD4 to enter cells. Consequently the viral envelope CD4-binding-site (CD4bs) is relatively conserved, making it a logical neutralizing antibody target. It is important to understand how CD4binding site variation allows for escape from neutralizing antibodies. Alanine scanning mutagenesis identifies residues in antigenic sites, whereas escape mutant selectionI dentifies viable mutants. We selected HIV-1 to escape CD4bs neutralizing MAbs b12, A12 and HJ16. Viruses that escape from A12- and b12- remained susceptible to HJ16, VRC01 and J3, whilst six different viruses that escape HJ16 remained sensitive to A12, b12 and J3. In contrast, their sensitivity to VRC01 was variable. Triple HJ16/A12/b12resistant virus proved that HIV-1 can escape multiple BNMAbs, but still retain sensitivity to VRC01 and the llama derived J3 nanobody. This antigenic variability could reflect that occurring in circulating viruses so studies like this could predict immunologically relevant antigenic forms of the CD4bs for inclusion in HIV-1 vaccines.
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CD4 binding site broadly neutralizing antibody selection of HIV-1 escape mutants
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Hanna Dreja1, Corinna Pade1, Lei Chen2, Áine McKnight*1
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1
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Centre for Immunology and Infectious Disease, Blizard Institute, Barts & The London School of Medicine and Dentistry, Queen Mary University of London 2
Vaccine Research Center, NIAID, National Institutes of Health, Bethesda, Maryland
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Running Title: Neutralizing antibody selection of HIV-1 escape mutants
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*Corresponding Author: Áine McKnight email [email protected]
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Words 2229
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Abstract
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All human-immunodeficiency-virus type-1 (HIV-1) viruses use CD4 to enter cells. Consequently the
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viral envelope CD4-binding-site (CD4bs) is relatively conserved, making it a logical neutralizing
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antibody target. It is important to understand how CD4-binding site variation allows for escape
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from neutralizing antibodies. Alanine scanning mutagenesis identifies residues in antigenic sites,
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whereas escape mutant selection identifies viable mutants. We selected HIV-1 to escape CD4bs
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neutralizing MAbs b12, A12 and HJ16. Viruses that escape from A12- and b12- remained
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susceptible to HJ16, VRC01 and J3, whilst six different viruses that escape HJ16 remained sensitive
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to A12, b12 and J3. In contrast, their sensitivity to VRC01 was variable. Triple HJ16/A12/b12-
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resistant virus proved that HIV-1 can escape multiple BNMAbs, but still retain sensitivity to VRC01
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and the llama derived J3 nanobody. This antigenic variability could reflect that occurring in
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circulating viruses so studies like this could predict immunologically relevant antigenic forms of the
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CD4bs for inclusion in HIV-1 vaccines.
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A successful human immunodeficiency virus type 1 (HIV-1) vaccine is expected to need to induce
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robust CD4+ and CD8+ cellular responses, in concert with a strong and broadly neutralizing
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antibody response. Designing immunogens that trigger such responses is challenging (reviewed in
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(Haynes & Montefiori, 2006; McCoy & Weiss, 2013)), partly due to the diversity (Gaschen et al.,
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2002) of the viral envelope glycoprotein (Env), which interacts with cell receptors including CD4.
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Hence, the CD4 binding site (CD4bs) is functionally conserved and is therefore a logical vaccine
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target to elicit neutralizing Abs. A number of highly effective anti-CD4bs broadly neutralizing
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monoclonal antibodies (BNMAbs) with distinct neutralization profiles have been generated from
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HIV-1 infected individuals (Burton et al., 1991; Burton et al., 1994; Corti et al., 2010; Falkowska et
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al., 2012; Walker et al., 2009; Wu et al., 2010; Zhou et al., 2010) or vaccinated llamas (Forsman et
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al., 2008; McCoy et al., 2012). A full understanding of the nature of these antibodies would
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provide clues to the antigenic landscape of the CD4bs, which may be important for developing an
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inclusive HIV-1 vaccine. In addition to structurally define current BNMAbs and their corresponding
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CD4bs footprints, the ability of replication competent viruses to escape such antibodies and to
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determine whether the mutants remain susceptible to alternative anti-CD4bs BNMAbs will be
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valuable.
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We used the three different anti-CD4bs BNMAbs b12, A12 and HJ16 to select for escape mutants.
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B12 was the first human BNMAb to map to the CD4bs and competes for binding to soluble CD4
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(sCD4) (Barbas et al., 1992; Burton et al., 1991; Burton et al., 1994; Roben et al., 1994; Zhou et al.,
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2010). The llama-derived single chain Ab A12 also competes with b12 and sCD4 (Forsman et al.,
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2008), as does HJ16 (Corti et al., 2010). Alanine scanning has determined that HJ16 belongs to a
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class of antibodies, which binds to a region distinct from the classic CD4bs (AA 474-476) (Pietzsch
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et al., 2010). Recently, Balla-Jhagjhoorsingh et al (Balla-Jhagjhoorsingh et al., 2013) identified a
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glycosylation site (N276) critical for HJ16-induced escape of a primary HIV-1 strain in an in vivo
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model.
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We used the well described HIV-1 replication competent clone HXB2 (Ratner et al., 1985) as it is
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unlike primary isolates highly sensitive to many anti-CD4bs BNMAbs. This is probably because
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HXB2 was highly passaged in vitro in the absence of humoral responses Escape viruses were
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selected in C8166 CD4+ T-cells (Salahuddin et al., 1983) in gradually increasing concentrations of
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BNMAbs (from 50 ng NMAb ml-1). Cell free supernatants were harvested from cells with cytopathic
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appearances and added to target cells for a second round of infection, this time with a doubling of
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the BNMAb concentration. After two to four weeks, resistant viruses emerged that could replicate
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in the presence of high concentrations of each BNMAb (10 µg A12 or HJ16 ml–1 and 20 µg b12 ml–
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1
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resistant viruses were PCR amplified and sequenced (Dreja et al., 2010).
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Selection of HXB2 with b12 resulted in a virus with the single dominant AA change (G366E) within
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the envelope CD4bs (Fig 1, 3). This change is located three AAs upstream of the proline to lysine
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mutation seen in a previous escape study (Mo et al, 1997), where additionally two mutations were
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observed in the V2 region. Interestingly, we did not identify any additional changes. This lack of
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other mutations within the V2 could be explained by the ease of neutralisation of HXB2,
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suggesting that supplementary V2 compensatory mutations were not required for resistance. It
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may be that the G366E mutation did not hamper viral infectivity. Glycine 366 has previously been
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implicated in b12 binding (Li et al., 2011; Saphire et al., 2001, Zhou et al., 2007). The site is
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mapped to a model of the crystal structure of the trimeric HIV-1 Env spike, (Fig 3). The HIV-1 viral
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env-expression vector psvIII-HXB2 (Gao et al., 1996) was engineered by site-directed mutagenesis
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(SDM) to carry the G366E mutation. SDM-pseudotyped virions carrying the luciferase reporter
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gene were produced as previously described (Dreja et al., 2010). The resulting SDM(b12)
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confirmed the b12 resistant phenotype (Fig 2a). The mutation had no effect on viral susceptibility
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to A12 and HJ16, even though both these BNMAbs compete for binding with b12, with each other
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and with sCD4. Moreover the sensitivity of SDM(b12) to VRC01 (Wu et al., 2010; Zhou et al., 2010)
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remained the same (Fig 2b). The recently described llama derived anti-CD4 BNMAb J3 (McCoy et
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al., 2012) neutralized SDM(b12) as efficiently as wild type (WT)-HXB2 (Fig 2b). These results
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suggest that the AA change responsible for b12 escape is antigenically distinct from those of the
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human BNMAbs HJ16 and VRC01 and the llama BNMAbs A12 and J3. The viral escape from b12
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had little effect on the ability of virus to replicate in vitro (data not shown) but was measurably
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less sensitive to CD4-IgG2 inhibition (IC50 shifted from 5ng ml-1 to 50 ng ml-1 and for the
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pseudotype SDM(b12) from 10ng ml-1 to 100 ng ml-1, Fig 2b).
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The A12-selected escape virus was resistant to A12 and carried the S375N mutation adjacent to
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the CD4bs (Fig 1 and 3). SDM(A12) confirmed that A12 resistance is conferred entirely by this
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mutation (Fig 2a). SDM(A12) remained sensitive to both b12 and HJ16, supporting the notion that
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the epitopes of A12 and b12 or HJ16 are distinct. The J3 llama antibody neutralized SDM(A12) and
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there is a small reduction in sensitivity to VRC01 for SDM(A12). There was, however, no apparent
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effect on the sensitivity of the mutation in SDM(A12) to CD4-IgG2.
). Neutralization assays were carried out and proviral full length env from cells infected with
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In contrast to the b12 and A12 selection, where dominant genotypes were generated, selection
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with HJ16 yielded a viral swarm, containing several different mutant viruses. It could be that there
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are more options for escape routes with this single chain antibody compared to bivalent
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antibodies which may have more steric hindrance. The proviral env sequences amplified from HJ16
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viral selected cultures (from nucleotide 127 (KpnI) to 2251 (BamHI)) were inserted into the env-
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expression vector psvIII-HXB2 and six different infectious, resistant clones were identified.
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Interestingly, and in keeping with the observation that glycosylation may be associated with HJ16
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resistance in primary cell cultures (Balla-Jhagjhoorsingh et al., 2013), we also observed that four
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mutations of a potential N-linked glycosylation site in the V5 loop affected sensitivity to HJ16
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neutralization (N463S, S465F, S465P and S465Y) (Fig 1, 3). Gray et al, (Gray et al., 2011)
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demonstrated a relatively high degree of sequence variation within the V5 loop in a large,
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independent panel of Envs, which may affect the accessibility to the CD4bs. Remarkably, none of
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these four substitutions significantly affected CD4-IgG2 inhibition (Fig 2b). The mutations are
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eleven and nine AAs upstream of the core region (474-476), identified as a HJ16 target by Pietzsch
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et al (Pietzsch et al., 2010). Curiously, of the three HJ16 resistant pseudoviruses with substitutions
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at position 465, two (psHJ16(S465F) and psHJ16(S465P)) gained sensitivity to VCR01 (Fig 2a). This
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is concurrent with alanine substitution of this residue (Falkowska et al., 2012), which increased
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sensitivity to VRC01 neutralization. In contrast, psHJ16(S456Y) retained wildtype sensitivity to
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VRC01. This suggests that the glycosylation per se is not important for the antibody footprint of
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VRC01, although it appears crucial for HJ16 activity. Similarly, psHJ16(N463S) maintained wildtype
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sensitive to VRC01. Overall our results suggest that the V5 region is involved in HJ16 and VRC01
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binding, as changes in this domain affect neutralization to both BNMAbs. The fifth HJ16 resistant
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virus had a glycine to aspartic acid change at position 459 (psHJ16D(G459D)), and resulted in a
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virus that was marginally more resistant to VRC01. This mutation was identified in HIV-1 (JRCSF)
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infected humanised mice treated with 45-46G54W, a BNMAb belonging to the VRC01 family (Klein
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et al, 2012). The G459D mutation is only four AAs upstream of the glycosylation site in the V5 loop,
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and exhibits a similar neutralisation profile as psHJ16(N463S) . By contrast, similar to S465F and
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S465P, the HJ16 resistant clone E409R also became more neutralization sensitive to VRC01 (Fig
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2b). All HJ16 resistant pseudotyped viruses retained sensitivity to J3, b12, CD4-IgG2 and, in four
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cases, to A12. Interestingly, G459D and E409R appeared more sensitive to A12 neutralisation at
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lower concentrations (1000 ng x mL-1). This partial VRC01 resistance is in accordance with the findings from
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the individual AA changes, where the N463S and G366E mutations were neutral, whereas S375N
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renders the virus less sensitive to VRC01 neutralization (Fig 2b). SDM(HJ16/A12/b12) was
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marginally less sensitive to J3 (IC50 from 3 to 25 ng x mL-1), as predicted by the lower sensitivity of
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the S375N and G366E mutations (Fig 2b). As expected, the sensitivity to CD4-IgG was partly lost,
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which is likely to be due to the G366E mutation described above (IC50 from 2 to 20 ng x mL-1).
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Nevertheless, it is intriguing that albeit marginally resistant to the BNMAbs VRC01 and J3, the
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triply selected virus retains some sensitivity to these BNMAbs. This suggests that if one succeeds in
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inducing a range of different anti-CD4bs Abs by vaccination, neutralization control of HIV-1 may be
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achieved. Also, the three BNMAbs, provided at the same time, never enabled for a resistant virus
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to emerge, cautiously suggesting that there is a limit to the amount of pressure the virus can
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withstand. Pre-exposure prophylaxis using modified BNMAb is considered as an option to prevent
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HIV-1 acquisition (Pace et al., 2013). Our findings would support such an approach and suggest
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that multiple BNMAbs targeting the CD4bs structure should be considered.
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Studies of the antigenic landscape of BNMAbs and the escape routes viruses master can help us to
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target important immunogenic epitopes for HIV-1 vaccines, but also to consider including
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anticipated escape structures.
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Authors' contributions
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HD and AM conceived and designed the experiments. HD and CP acquired the data. HD and AM
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interpreted the data and drafted the manuscript. LC mapped mutations onto HIV-1 trimer and
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provided structural context to data. All authors read and approved the final manuscript.
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Acknowledgements
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B12 was provided by Dennis Burton, The Scripps Research Institute; A12 and J3 from Robin Weiss,
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UCL; HJ16 from Humabs Biomed SA, Switzerland. VRC01 was obtained through the AIDS Research
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and Reference Reagent Program, Division of AIDS, NIAID, NIH, from John Mascola and CD4-IgG2
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through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Cat#11780 from Progenics
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Pharmaceuticals. The genetic replication competent clone HXB2 was obtained from the Centre for
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AIDS Research (NIBSC, UK). psvIII-HXB2 was provided by Paul Clapham, Worcester, MA. This
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research was conducted as part of the Collaboration for AIDS Vaccine Discovery funded by the Bill
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and Melinda Gates Foundation (Weiss VDAC, UCL). We would like to thank Robin Weiss, Peter
206
Kwong and Laura McCoy for critically reading the manuscript.
207
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Figure legends
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Figure 1
310 311
Sequences of the BNMAbs-selected EMs are compared to the parental HXB2 env gene, with the nucleotide number in italics to the left. EM3 is the HJ16/A12/b12 triply selected virus.
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Figure 2
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HXB2 (white discs), SDM(A12), SDM(b12) and HJ16-resistant pseudotyped HIV-1 virions (black discs) were assessed for neutralization resistance against against (a) A12, b12 and HJ16 and (b) VRC01, J3 and CD4-IgG2. The % of neutralization is shown on the y-axis in the presence of BNMAbs (in bold at the bottom of the graph) at different concentrations (x-axis: ng x ml-1).
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Figure 3
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A model of the crystal structure of the HIV-1 Env trimer, where identified AA changes are indicated with arrows. HXB2 gp120 is adapted from 3JWD (Pancera et al., 2010). The trimer is adapted from 4NCO.
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Figure Click here to download Figure: Dreja 10 Feb2015.pptx
Figure 1
HXB2
360 FKQS SGGDPE IV THSFNCGG E FFYCNSTQL FNSTWFNST W STEGSNNT EG SD TITLPCRI KQIIN MWQKV GKA MYAPPIS
GQ IRCSSNIT G LLLTRDGGN SNNESEIFR P
EM(b12)
---- -E---- -- -------- - --------- --------- - -------- -- -- -------- ----- ----- --- ------- -- -------- - --------- --------- -
EM(A12)
---- ------ -- --N----- - --------- --------- - -------- -- -- -------- ----- ----- --- ------- -- -------- - --------- --------- -
EM(HJ16F) ---- ------ -- -------- - --------- --------- - -------- -- -- -------- ----- ----- --- ------- -- -------- - --------- ----F---- EM(HJ16P) ---- ------ -- -------- - --------- --------- - -------- -- -- -------- ----- ----- --- ------- -- -------- - --------- ----P---- EM(HJ16Y) ---- ------ -- -------- - --------- --------- - -------- -- -- -------- ----- ----- --- ------- -- -------- - --------- ----Y---- EM(HJ16S) ---- ------ -- -------- - --------- --------- - -------- -- -- -------- ----- ----- --- ------- -- -------- - --------- --S------ EM(HJ16D) ---- ------ -- -------- - --------- --------- - -------- -- -- -------- ----- ----- --- ------- -- -------- - -------D- --------- -
EM(HJ16R) ---- ------ -- -------- - --------- --------- - -------- R- -- -------- ----- ----- --- ------- -- -------- - --------- --------- EM3
---- -E---- -- --N----- - --------- --------- - -------- -- -- -------- ----- ----- --- ------- -- -------- - --------- --S------ -
psHJ16 (S465Y)
psHJ16 (N463S)
psHJ16 (G459D)
psHJ16 (E409R)
% neutralization % neutralization % neutralization
psHJ16 (S465P)
% neutralization
psHJ16F (S465F)
% neutralization
SDM(b12) G366E
% neutralization
WT HXB2
% neutralization
SDM(A12) S375N
% neutralization
Figure 2A 120 100 80 60 40 20 0 120 100 80 60 40 20 0 120 100 80 60 40 20 0 120 100 80 60 40 20 0 120 100 80 60 40 20 0 120 100 80 60 40 20 0 120 100 80 60 40 20 0 -20 120 100 80 60 40 20 0 -20
A12
1
10
1
10
1
1
1
1
1
1
b12 120 100 80 60 40 20 0 120 100 1000 100 80 60 40 20 0 100 1000 120
100 80 60 40 20 0 120 10 100 1000 100 80 60 40 20 0 120 10 100 1000 100 80 60 40 20 0 10 100 1000120 100 80 60 40 20 0 10 100 1000 120 100 80 60 40 20 0 10 100 1000120 100 80 60 40 20 ng x ml-1 0 10
100
1000
1
10
1
10
1
10
1
10
1
10
1
10
1
120 100 80 60 40 20 0 100 1000 120 100 80 60 40 20 0 100 1000 120 100 80 60 40 20 0 120 100 1000 100 80 60 40 20 0 100 1000 120 100 80 60 40 20 0 100 1000 120 100 80 60 40 20 0 -20 100 1000 120
10 100 1000
ng x ml-1 1
10
HJ16
100 1000
100 80 60 40 20 0 -20 120 100 80 60 40 20 0 -20
1
10 100 1000
1
10 100 1000
1
10 100 1000
1
10
100 1000
1
10
100 1000
1
10
100 1000
1
10
100 1000
ng x ml-1 1
10
100
1000
psHJ16 (S465Y)
psHJ16 (N463S)
psHJ16 (G459D)
psHJ16 (E409R)
120 100 80 60 40 20 0 120 1 10 100 1000 100 80 60 40 20 0 120 -20 1 10 100 1000 100 80 60 40 20 0120 -20100 1 10 100 1000 80 60 40 20 0 -20 1 10 100 1000 120 100 80 60 40 20 0 -20 1 10 100 1000 120 100 80 60 40 20 0 -20 1 10 100 1000 120 100 80 60 40 20 ng x ml-1 0 1 10 100 1000 -20
J3
120 100 80 60 40 20 0
120 100 80 60 40 20 0 120 100 80 60 40 20 0 120 100 80 60 40 20 0
120 100 80 60 40 20 0
120 100 80 60 40 20 0
120 100 80 60 40 20 0
120 100 80 60 40 20 0
120 100 80 60 40 20 0 120 100 80 60 40 20 0
120 100 80 60 40 20 0
% neutralization
% neutralization
120 100 80 60 40 20 0 120 100 80 60 40 20 0
% neutralization
psHJ16 (S465P)
VRC01
% neutralization
psHJ16F (S465F)
120 100 80 60 40 20 0
% neutralization
SDM(b12) G366E
% neutralization.
WT HXB2
% neutralization
SDM(A12) S375N
% neutralization
Figure 2B
1
10
120 100 80 60 40 20 0
100 1000
ng x ml-1 1
10
100 1000
CD4-IgG2
1
10 100 1000
1
10 100 1000
120 100 80 60 40 20 120 100 80 60 40 20 0 -20
ng x ml-1 1
10
100 1000
Figure 3
HJ16: N463S HJ16: G459D HJ16: E409R
A12: S375N
b12: G366E
gp41
HJ16: N463S HJ16: G459D HJ16: E409R b12: G366E
Glycan gp120s
CD4BS A12: S375N
